Objective To construct AWP1 (associated with protein kinase C related kinase 1) recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304. Methods Cloned AWP1 cDNA was inserted into the multiply clone sites (MCS) of plasmid pcDNA3 for adding flag tag, and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV. After identified with restrictional enzymes, plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease PmeⅠ, and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination. After linearized by PacⅠ, the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high-level adenoviruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope (LSCM). Results PCR assay showed that recombinant adenovirus Ad-flag-AWP1 was obtained successfully; and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWP1 recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.
Objective To study and summarize the clinical experience and significance of the skeleton reconstruction of human hand allografts. Methods From January 2001 to October 2003, human hand allografts were appliedto treat 4 cases of traumatic hand defect(6 hands) at different levels. During operation, the ulna and radius were reduced anatomically and fixed firmly with 3.5 mm AO-plates and screws according to AO internal fixation principle. The X-ray films were taken periodically andthe function recovery of hand allografts was observed and estimated. Results The 4 cases were followed up for 4-36 months postoperatively. The clinical healing of fracture in 4 cases(6 hands) was achieved after 9 weeks,and by means of comprehensive assessment including the joint function, muscle strength, sensation, appearance, sequela and the ability of work, the satisfactory effects were gained eventually. Conclusion It is significant forhuman hand allografts to reconstruct skeleton firmly.
【Abstract】 Objective To reconstruct three different kinds of tympanic membrane in vitro by tissue engineeringtechnique, and to examine their histological structures and mechanical properties. Methods The skin and dura of pig(weight 30 kg) were processed with high satuated sal ine and enzymes to make extracellular matrix. Meanwhile, fibroblasts(1×106 /mL,0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties. Results Fibroblasts cultured were spindle–shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibil ity. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 μm respectively. The tension of the collagen was (1.417±0.030) N/mm2, of the acellular dermal matrix was (24.500±2.040) N/mm2(being close to the tension of normal tympanic membrane, 26.700 N/ mm2),of the acellular dura was (53.300±2.600) N/mm2. Conclusion The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.
Objective To summarize the recent progress of construction methods of engineered cell sheet and to forecast the possible prospect. Methods The recent original articles about investigation and appl ication of engineered cell sheet were reviewed. Several common methods were selected and expounded. Results The construction methods of engineered cell sheet mainly include temperature-responsive culture dish, salmon atelocollagen, magnetic force, surface roughness, and polyelectrolytes, which may overcome the l imits of traditional tissue engineering methods. Conclusion The construction methods of engineered cell sheet are feasible and have a bright future in the cl inical appl ication.
ObjectiveTo construct a framework and functional items of a scientific research assistant tool for conducting systematic review for patient-reported outcome measures. MethodsBased on the research foundation and work experience of the system evaluation of two patient-reported outcome measures systematic reviews carried out by the research group in the early stage, the framework and function system of scientific research aid tool was initially constructed, and two rounds of correspondence were carried out by Dephi expert consultation method. ResultsThe effective recovery rates of the two rounds of expert consultation questionnaires were 90% and 100%, the expert authority coefficient was 0.839, and the compatibility coefficients of suitability and importance were 0.105 and 0.177, respectively. The final the patient-reported outcome measures tool system evaluation scientific research aid tool system consists of 7 frames and 31 items. ConclusionThis study has developed a scientific and comprehensive set of functional criteria for research-assistant tools that systematically review patient-reported outcome measures based on the COSMIN methodology and it lays the foundation for subsequent tool research and development.
Objective To study the interferencing and anti-tumor effects of lentiviral vector of siRNA targeting IGF1R and EGFR gene of the liver cancer cell. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and connected to the pLVTHM vector, named pLVTHM-IGF1R, into whom the EGFR-siRNA expression frame containing H1 promotor synthesized by RT-PCR was cloned to generate pLVTHM-IGF1R-EGFR-siRNA. The 293T cells were cotransfected by 3 plasmids of pLVTHM-IGF1R-EGFR-siRNA, psPAX2 and pMD2G to enclose LVTHM-IGF1R-EGFR-siRNA, which was amplified in large amount and purified by caesium chloride density gradient centrifugation for measurement of virus titer. SMMC7721 cells infected by LVTHM-IGF1R-EGFR-siRNA were infection group, the untreated SMMC7721 cells and blank vector plasmid LVTHM were two control groups (SMMC7721 cell group and blank vector group). The effect of LVTHM-IGF1R-EGFR-siRNA on IGF1R and EGFR expressions of SMMC7721 cells were detected by RT-PCR and Western blot. The antitumor potential of LVTHM-IGF1R-EGFR-siRNA to SMMC7721 cells was evaluated by Cell Counting Kit-8 assay for cell growth and TUNEL for apoptosis respectively. Results LVTHM-IGF1R-EGFR-siRNA was constructed successfully. Functional pfu titers of LVTHM-IGF1R-EGFR-siRNA was 4.58×109 pfu/ml. Protein and mRNA expression of IGF1R and EGFR of infection group were less than those of blank vector group and SMMC7721 cell group （P＜0.05）, LVTHM-IGF1R-EGFR-siRNA was more effective to inhibit the proliferation and promote apoptosis of SMMC7721 cells （P＜0.05）. Conclusion LVTHM-IGF1R-EGFR-siRNA expressing IGF1R-EGFR-siRNA can inhibit the expression of IGF1R and EGFR, and may be used for further investigation of gene therapy of liver cancer.
Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
ObjectiveTo explore the current situation of financial toxicity (FT) of breast cancer patients undergoing daytime chemotherapy under the background of diagnosis intervention packet (DIP) and its influencing factors, and to build a risk early warning model.Methods Convenient sampling method was used to select breast cancer patients undergoing chemotherapy in the daytime ward of Tianjin Medical University Cancer Institute & Hospital between April and May 2022. The general data questionnaire and FT comprehensive score scale were used to investigate them, and the influencing factors of patients’ FT were discussed through single factor analysis and logistic regression analysis, and the risk early warning model was established. Hosmer-Lemeshow fitting effect test was used to evaluate the prediction effect of the model.Results A total of 278 patients were included. The median (lower quartile, upper quartile) of FT score was 14.00 (8.75, 23.00), of which 195 patients (70.14%) had FT score≤22; 83 patients (29.86%) had FT scores>22. Logistic regression analysis showed that age, per capita monthly income of families, commercial health insurance, chemotherapy cycle, tumor stage, neoadjuvant chemotherapy were the influencing factors for high-risk FT of breast cancer patients undergoing daytime chemotherapy. The results of Hosmer-Lemeshow goodness of fit test showed that the model-predicted FT of breast cancer patients undergoing daytime chemotherapy was in good agreement with the actual observation value (χ2=10.685, P=0.220). The area under the curve of the model was 0.931 [95% confidence interval (0.900, 0.962)], the sensitivity was 0.807, and the specificity was 0.913.Conclusions The FT of breast cancer patients undergoing daytime chemotherapy is at a high level. Older age, purchase of commercial health insurance, and high per capita monthly income of families are protective factors for high-risk FT. The wind with chemotherapy cycle≤4 weeks, tumor stage Ⅱ, neoadjuvant chemotherapy are high-risk FT risk factors. The final warning model has been tested to have a good prediction effect, which can provide a reference for clinical medical staff to identify high-risk FT patients early and make preventive strategies as soon as possible.
ObjectiveTo evaluate the developing methodologies of Essential Medicines Lists for Children (EMLcs) in global, in order to provide reference in developing EMLc of China. MethodsWe searched ProQuest, ScienceDirect, SpringerLink and MEDLINE databases, World Health Organization (WHO) official website, and 67 websites of National Ministry of Health and Drug Administration Section, to collect literature about selection methodology of children and/or adult essential medicines list (EML). A descriptive analysis was conducted. ResultsA total of fourteen literatures were included. Of which, 6 were about the essential medicines selection methodology in children, and the other 8 were about the essential medicines selection methodology in adult. The WHO had established independent EMLc selection committee. Paediatricians were involved in the selection of EMLc in the WHO and India. There was no selection criteria and process for EMLc globally. The WHO, India, and South Africa selected their EMLcs referring to the WHO EML selection criteria. The WHO and South Africa had their own updating time, period and process for EMLc. The WHO EMLc was updated per 2 years, which in high frequency and conducts in rigorous process. However, the EMLc of India had not been updated yet. ConclusionIt is suggested that China could build a national EMLc selection committee involving paediatricians and evidence-based medicine experts etc. in referring to the framework of the WHO Child Health Working Group. The EMLc selection criteria and process of China could be established referring to the one of the WHO, based on the disease burden, drug accessibility and medical insurance of children of China. The EMLc of China should be simultaneously updated with the adult EML of China.
Objective To search the most suitable concentration of calcium in the medium for the basement membrane reconstruction in tissue engineering skin in vitro. Methods Composite chitosan tissue engineering skin was prepared according to previous studies. Four groups were included according to the concentrationof calcium (1.00, 1.45, 1.65 and 1.95 mmol/L respectively). After 7 days and 15 days of culture, the histological manifestation of basement membrane in tissue engineering skin was observed by hematoxylin amp; eosin staining and PAS staining, and collagen Ⅳ of basement membrane was detected immunohistochemicallyatthe dermalepidermal junction. Results This tissue engineering skin shared some histological features of normal skin, including a welldifferentiated stratifiedepidermis and a dense dermis. The epithelium in the group of 1.95 mmol/L calcium differentiated better than those in other groups. PAS staining showing a regularly red dying strap domain at the dermal-epidermal junction. Collagen Ⅳ was positively stained immunohistochemically at the dermalepidermal junction inthe tissue engineering skin. Conclusion The above results suggest that the medium with 1.95 mmol/L calcium should be suitable for the growth of composite chitosan tissue engineering skin and the reconstruction of basement membrane.